Prediction and detection of ovulation are critical steps in regulating female fertility and in aiding family planning. To this end, a variety of methods have been proposed in the prior art such as, for example, daily assay of the serum or urine for the presence of leutenizing hormone (LH), estrogens, progesterone, and the like. Such techniques, however, are cumbersome, expensive, and require the services of a skilled technician.
The daily recording of basal body temperature (BBT) is a well known method for detecting ovulation. Considerable skill, however, is required in observing and interpreting the data; furthermore, the BBT record does not predict the day of ovulation but rather provides evidence of ovulation two or three days after it has occurred.
Increases in peroxidase activity in the saliva, vaginal fluids, and cervical mucus have been measured as a means for detecting ovulation. Foster, R. O., U.S. Pat. No. 3,472,738 discloses a test for measuring the increase in peroxidase activity in the saliva around the time of ovulation which involves mixing the saliva with guaiacol, hydrogen peroxide, and sodium pyruvate to produce a blue color. More recently, measurement of salivary peroxidases whose activity increases around ovulation has been described by Cockle, S. M. et al., Brit. J. Obstet. Gyneacol. 85, 776-82 (1978) and Tenovuo, J. et al., Biochem. Med. 25, 337-345 (1981).
Vaginal fluids have been reported to react with hydrogen peroxide and a variety of compounds such as aromatic amines, guaiacol, and dyes to produce a blue color which increases in intensity around ovulation (Oster G. et al., International Application No. PCT/US 80/00618).
Blain J. A. et al., Contraception 11, 677-680 (1975) describe a procedure for measuring G-P.sub.x levels in cervical mucus which involves mixing a mucus sample with a phosphate-citrate buffer at pH 6.5 and with relatively large amounts of hydrogen perioxide and guaiacol. G-P.sub.x content of the cervical mucus was measured throughout the menstrual cycle and was found to be at a low level with no change occurring until the final week. The procedure showed no decrease in G-P.sub.x level at the point of incipient ovulation.
Additional techniques for predicting and detecting ovulation are discussed by Moghissi, K. S. Fetility and Sterility, 34, No. 2, 89-98, August 1980, which citation is incorporated herein by reference as illustrative of the state of the art. Moghissi concludes that "Currently no reliable method for prediction of ovulation is available, but a better understanding of cyclic changes in the constituents of cervical mucus and in the pattern of excretion of sex hormone metabolites may lead to the development of improved methods of ovulation detection as well as ovulation prediction."
Accordingly, it is an object of this invention to provide a reliable method for predicting ovulation in the female species based on an assay of G-P.sub.x in cervical mucus or vaginal fluids.
It is another object of this invention to provide a simple test for G-P.sub.x levels in cervical mucus or vaginal fluids that can be easily practiced by a woman at home to provide her with reliable means for natural family planning.
It is still another object of this invention to provide a simple kit for carrying out the inventive test method. These and other objects will become apparent as description of the invention proceeds.
In accordance with this invention, a method for detecting impending ovulation in human female comprises an assay for the decrease in G-P.sub.x levels in cervical mucus at the time of impending ovulation. In the method, a sample of cervical mucus obtained from either the cervical os or the vaginal canal is extracted with an aqueous solution comprising Ca.sup.++ and a buffer providing a slightly alkaline pH. The extract is admixed with a small amount of guaiacol and peroxide whereupon the presence of G-P.sub.x is indicated by the development of color having maximum absorbance at 470 nm. Daily assays are performed until an abrupt color change signals a significant drop in G-P.sub.x level and the onset of the ovulation process. Although decrease in G-P.sub.x levels coincident with impending ovulation is assayed preferably in samples of cervical mucus, it is understood that the test procedure is applicable to assaying the corresponding changes in salivary peroxidase levels.
A suitable solution for extraction of G-P.sub.x comprises a water soluble calcium salt and a buffer that is non-reactive with Ca.sup.++ and that provides a slightly alkaline pH. A solution that can be employed to advantage contains 0.5M CaCl.sub.2 and 10 mM Tris-Cl buffer at a pH of about 7.2.
Peroxides that can be used include hydrogen peroxide, sodium peroxide, barium peroxide, sodium perborate, and the like. Hydrogen peroxide is generally preferred because of its ready availability, complete water solubility, and ease of concentration control.
For precise clinical assay, changes in G-P.sub.x levels during the menstrual cycle can be followed by absorbance measurements at 470 nm with a spectrophotometer. The decrease in G-P.sub.x levels from about 1000 to 100 to 1 units is associated with a visual color change from deep red to pink to yellow. A level of about 100 units or a color change from deep red to pink indicates impending ovulation and the onset of the fertile period. That period of the cycle is ended when the assay again shows a deep red color.